Effect of Different Mixing Methods on Physicochemical Properties of Mineral Trioxide Aggregate: A Systematic Review

Background Mineral trioxide aggregate (MTA) is a commonly used endodontic biomaterial. The physicochemical properties of MTA have a crucial role in designating clinical outcome, and different factors can affect these properties. Various methods have been used for mixing MTA, including manual, mechanical, and ultrasonic. The aim of this systematic review was to evaluate the effect of different mixing methods on the physicochemical properties of MTA. Materials and Methods Electronic databases including PubMed, Embase, Web of Science, and Scopus were searched up to May 2022. In order to cover gray literature, the ProQuest and Google Scholar databases were also searched to detect theses and conference proceedings. For quality assessment of the included studies, we used a modified version of the Cochrane risk of bias tool for randomized controlled trials (RCTs). Experimental studies which had assessed at least one property of MTA and compared at least two different mixing methods of MTA were included in this study. All animal studies, reviews, case reports, and case series were excluded. Results Fourteen studies were included. The results showed that the ultrasonic mixing method significantly improved some MTA characteristics, including microhardness, flowability, solubility, setting time, and porosity. However, the mechanical mixing method improved other properties including flowability, solubility, push-out bond strength, and hydration. The manual mixing method showed inferior results compared to other mixing methods in terms of microhardness, flowability, solubility, setting time, push-out bond strength, porosity, and hydration. Different mixing methods had a similar effect on compressive strength, sealing ability, pH and calcium ion release, volume change, film thickness, and flexural strength of MTA. Conclusion Mechanical and ultrasonic mixing methods are superior to the manual mixing method in terms of improving physicochemical properties of MTA. No report of selection bias and varieties in methodologies were limitations of evidence.


Introduction
Te role of bioactive materials in dentistry is undeniable. Increasing the usage time of restoration, stimulating the dentin repair process, and favoring adhesive resistance are all positive efects of bioactive materials [1][2][3]. One of the well-known bioactive materials in the endodontics fled is including long setting time and difcult handling [12,13]. Its applications in endodontics are pulp capping, perforation repair, apexifcation, pulpotomy [14,15], obturation, and apical plug [16,17].
Physicochemical properties of an endodontic biomaterial are crucial for their efective clinical use. To attain these ideal characteristics in hydraulic cement, the elements should be completely mixed with water. Tree mixing methods commonly used to mix MTA include manual, mechanical, and ultrasonic methods.
Many studies have tried to investigate the efect of diferent mixing methods on various characteristics of MTA with controversial results. For example, in a survey on fowability, volume change, solubility, and pH of MTA, Duque et al. showed that the mixing methods could not afect the fowability of MTA signifcantly [18]. However, Shahi et al. [19] showed that the mechanical and ultrasonic mixing methods had higher fowability than the manual technique. Te discrepancies in the results of studies may confuse the clinicians for choosing the appropriate way of mixing MTA to achieve optimum physicochemical characteristics. Terefore, this systematic review aimed to compare the efect of diferent mixing methods on the physicochemical properties of MTA to help clinicians choose the appropriate mixing method.

Study Design.
Tis systematic review was accomplished in agreement with the recommendations of preferred reporting items for systematic reviews and meta-analysis (PRISMA) (Supplementary Material 1) [20,21]. Table 1, the study question was "what are the efects of various MTA mixing methods (I) on the physicochemical properties (O) of MTA (P)?" Electronic databases including PubMed, Embase, Web of Science, and Scopus were searched up to May 2022. In order to cover gray literature, the ProQuest and Google Scholar databases were also searched to detect theses and conference proceedings. Backward and forward reference searching was also performed. Te search strategy for all databases is shown in Table 1.

Inclusion Criteria.
Te inclusion criteria were as follows: in-vitro studies assessing at least one physicochemical property of MTA published in English, and studies comparing at least two diferent mixing methods with defned sample sizes.

Exclusion Criteria.
All animal studies, reviews, case reports, and case series were excluded. Also, studies in which their language was not English were excluded.

Study Selection.
After removing duplicates, two authors (A.S.M and F.R) individually screened the titles and abstracts. Te full text of the remaining studies was read, and relevant studies according to the eligibility criteria were selected. A third author (M.B) resolved any disagreements between the reviewers.
2.6. Data Extraction. Two authors (M.B and F.R) individually extracted the following information from the studies: author(s), year of publication, types of specimens, mixing methods, time of assessment, assessment tools, assessed properties, and outcomes. Any disagreement regarding this process was resolved by a third author (B.R).

Quality Assessment.
For quality assessment of the included studies, a modifed Cochrane risk of bias tool was used [22,23]. Two independent reviewers (A.S.M and M.B), which were both dentists and comprehensively informed of the topic and the details of the Cochrane risk of bias tool according to published guidelines [24], checked the following biases: selection bias, detection bias, attrition bias, reporting bias, and other biases. Any disagreements were discussed with a third author (M.H) and resolved.

Assessment of Heterogeneity and Synthesis of Results.
Te heterogeneity of included studies regarding the mixing method, time of assessment, physicochemical characteristics, type of MTA, and the assessed properties and tests was examined.
Finally, 14 papers were included in the review ( Figure 1). Table 2 shows the results of the risk of bias assessment. Te risk of bias in the included studies showed low attrition and reporting bias (14/14 studies), followed by other biases (13/ 14 studies) and selection bias (7/14 studies) ( Table 2). Te extracted data are summarized in Table 3. All 14 included articles were experimental studies. Te summary of the risk of bias evaluation is shown in Figure 2. Te main source of bias in the included studies was the detection bias, which was unclear in all articles. Selection bias was also unclear in half of the included studies.
Heterogeneity of the included studies was high regarding the mixing method, time of assessment, physicochemical characteristics, type of MTA, and the assessed properties and tests. Te lack of univocal and standard experimental processes made a comparison of the results difcult; therefore, conducting a meta-analysis was not possible.

Microhardness.
Microhardness is an indicator of physical characteristics, such as yield strength, modulus of elasticity, tensile strength, and setting [48].
Nekoofar et al. [38] investigated the microhardness of four types of MTA (Angelus white, ProRoot grey, Angelus grey, and ProRoot white) mixed with manual, mechanical, and ultrasonic methods. Tey showed that irrespective of the type of MTA, the ultrasonic method showed the highest surface microhardness at 4 and 28 days compared with other techniques. Also, no signifcant diference was found between manual and mechanical methods [38]. Te authors attributed better results of the ultrasonic group to the dispersing efect that may provide enough space for water molecules and enhances water difusion resulting in a better degree of hydration and consequently a greater surface microhardness.
In another study, Saghiri et al. [43] investigated the microhardness of the white MTA mixed with manual, mechanical, and ultrasonic methods. Results showed that the mechanical mixing method had a signifcantly higher surface hardness compared to the other techniques [43]. Tey attributed these results to the needle-like crystals in MTA. Interestingly, they attributed the dispersing efect to inferior results obtained in the ultrasonic group. Also, the interaction of needle-like crystals of MTA may reduce the MTA microhardness through interlocking these crystals via ultrasonic energy. However, it should be noted that the growth of crystals takes place gradually after the mixing, and it is unclear how using ultrasonic energy for mixing MTA can afect the interaction of crystals. Terefore, conducting other studies with standard time intervals and various types of MTA cement is needed.

Flowability.
Flowability is the ability to penetrate the lateral and accessory canals and irregularities during canal obturation [49]. So, the fowability of the endodontic materials is a critical factor for high-quality obturation.
Shahi et al. [19] investigated the fowability of White ProRoot MTA mixed with manual, mechanical, and ultrasonic methods. Tey showed that the mechanical and ultrasonic mixing methods had higher fowability than the manual technique [19]. However, mechanical and ultrasonic techniques did not have any signifcant diference. In the second study, Duque et al. [18] showed that the fowability of MTA was not afected by the mixing technique [18].
Te diference between these studies can be attributed to the diferences in the details of manual mixing, the type, and the amount of MTA used.

Compressive Strength.
Te compressive strength is the ability to withstand heavy occlusal and restorative forces [50]. Te compressive strength of MTA is afected by factors such as the type of MTA, condensation pressure, mixing method, and the liquid mixed with MTA [8].
Shahi et al. [19] investigated the compressive strength of White ProRoot MTA mixed with manual, mechanical, and ultrasonic methods at two diferent time intervals (21 hours and 21 days). Tey showed that the efect of three diferent mixing methods on compressive strength was not signifcantly diferent at any time [19]. In another study, Basturk et al. [39] investigated the compressive strength of ProRoot MTA and MTA Angelus mixed with manual and mechanical mixing methods 4 days after mixing. Irrespective of the MTA type, the mechanical method showed higher compressive strength than the manual method [39]. No signifcant diference between the two mixing methods in both ProRoot and MTA Angelus was shown. Encapsulation alongside mechanical methods produced more homogeneous MTA slurries [38,41]. Tey assumed that better water difusion might be related to creating a less grainy mixture with fewer unhydrated particles in the mechanical method. Conversely, the manual method was associated with inadequate hydration by restraining the microchannel creation in the material and obstructing the entrance of water molecules to hydrate the material [38]. Tese conficting results may be due to diference in the type of MTA used and the time of assessment.

Te pH and Calcium Ion Release.
As one of the most important features of medical materials is biocompatibility [51], therefore, one of the superiorities of the MTA is its safe use in the dental canal [52]. Te biocompatibility of MTA is ascribed to its pH and calcium ion release [53].
Higher pH values are essential for the induction of hard tissue and antimicrobial properties [39,[54][55][56][57]. After mixing, the pH of MTA is 10.2 and increases to 12.5 at 3 hours. Te authors related the high pH to the continued release of calcium from MTA and the calcium hydroxide formation [58]. Shahi et al. [8] investigated the pH of MTA mixed with manual, mechanical, and ultrasonic methods at the end of the 1 st hour. Te pH was not signifcantly affected by diferent methods. In another study, Duque et al. [18] investigated the pH and the release of calcium ion of MTA Angelus mixed with manual, mechanical, and ultrasonic methods in four diferent time intervals (3, 24, 72, and 168 hours). Te mixing technique did not infuence pH values [18]. Te calcium ion release was higher with trituration compared to the manual technique at 3 and 168 hours [18].
Collectively, it could be concluded that diferent mixing methods of MTA did not have a statistically signifcant efect on pH and the calcium ion release.

4.5.
Solubility. Solubility is defned as the quantity of a solid material that can be dissolved in a certain amount of solvent. Variations in MTA solubility shown in diferent studies are due to such factors at the time of immersion, MTA type, and the powder-to-water proportion [13,[59][60][61]. Te low solubility means that the MTA remains where it has been placed, providing satisfactory flling and averting bacterial microleakage [62]. Most studies have suggested low or no solubility for MTA [62][63][64][65]. However, a long-term study reported a greater solubility [66]. Shahi et al. [8] investigated the solubility of MTA mixed with manual, mechanical, and ultrasonic methods. Te solubility was determined based on the modifed ADA guidelines No.30 and ISO 6876 by measuring the weight diference in three diferent time intervals (1, 7, and 21 days). Te mechanical and ultrasonic techniques resulted in higher solubility than the manual technique, though it was not statistically signifcant [8]. In another study, Duque et al. [18] investigated the solubility of MTA Angelus mixed with manual, mechanical, and ultrasonic methods. Te solubility was determined based on the modifed ADA specifcation 57 by measuring the weight diference at the end of day 7. Interestingly, they revealed that the sample's weight was increased over time. Te difference in MTA weights in the mechanical and ultrasonic methods was greater compared to the manual method [18].
In conclusion, the results of both studies showed that the weight change in the manual method was smaller than in the ultrasonic and mechanical methods. However, the authors had diferent interpretations of the weight change of samples, which could be related to the diferent methodologies used. Duque   Measuring weight before and after storage in water may not show real solubility since particles of the substance may detach from the cement in the stored environment, or the cement may absorb water. Such interactions seem to mislead investigators in the case of the evaluation of solubility [67,68]. Further normal saline can be used instead of distilled water to better simulate the physiologic condition of the MTA environment.

Initial and Final Setting Time (ST).
While the initial ST is defned as the time needed by the cement to set and to be rigid enough to bear the lighter Gillmore needle, the fnal ST is defned as the time necessary for the cement to support the heavier Gilmore needle with no signifcant indentation [69]. Te mixing method, quantity of water used, packing force, and moisture in the environment would afect the ST [70][71][72]. Although we found three articles conducted on setting and working time, we excluded one of them due to numerous problems in methods and results. In addition, it was not possible to reanalyze the results to fnd out the exact and correct results [44]. So, two articles about ST were included.
Duque et al. [18] investigated the initial and fnal ST of MTA Angelus mixed with manual, mechanical, and ultrasonic methods. In 60-second intervals, the mixing methods were not diferent regarding the initial and fnal ST of MTA [18].
In the second study, Saghiri et al. [43] investigated the initial STof white MTA mixed with those mixing methods in 60-or 300-second intervals and concluded that the ultrasonic technique signifcantly increased the initial ST compared to other techniques [43].
Both studies showed that manual and mechanical methods had not any signifcant efect on the initial ST of MTA. However, unlike Duque et al. [18], Saghiri et al. [43] exhibited that the ultrasonic technique signifcantly increased the initial ST. Because their methods were similar, this diference might be attributed to diferent types of MTA. Te only study measuring fnal ST showed no diference between diferent methods [18].

Film Tickness. Film thickness (FT) is assessed by
placing materials between the two glass slabs for few minutes after mixing based on ISO 6876 : 2001 specifcations [44]. Shahi et al. [44] investigated the FT of MTA Angelus mixed with manual, mechanical, and ultrasonic methods 10 minutes after mixing. Te mixing method did not infuence the FT of MTA [44].

Volume
Change. Less volume change during setting would be a favorable characteristic of MTA to assure its adaptation and prevent leakage. Minor expansion might be acceptable by improving the substance's adaptation. However, extreme volume change during the setting process may lead to microleakage, loss of marginal integrity, or fractures and cracks in the dental root [73].
Duque et al. [18] investigated the volume change of MTA Angelus mixed with manual, mechanical, and ultrasonic methods by volumetric micro-CT measurements and reported that at the 7 th and 14 th days of immersion, there was no signifcant association between the mixing method and the volume change [18]. In the second study, Shahi et al. [44] investigated the volume change of MTA Angelus mixed with manual, mechanical, and ultrasonic methods by digital Vernier measuring tool at the end of day 30, and they also reported that the volume change of MTA was not afected by the mixing technique [44]. Collectively, both studies confrmed that diferent methods did not have a signifcant efect on the volume change of MTA.

Push-Out Bond Strength.
One of the superior properties of MTA compared to other materials is the bonding ability to dentin and resistance against displacing forces [74]. Tus, the push-out strength is an important property of MTA as    Shahi et al. [41] investigated the push-out bond strength of a 72-hour set MTA Angelus mixed with manual, mechanical, and ultrasonic methods and reported that the mean push-out strength values of MTA by three diferent methods were similar [41]. In the second study, Uzunoglu et al. [45] investigated the push-out bond strength of ProRoot MTA mixed with manual and mechanical methods. Tey showed that the mechanical method had signifcantly higher bond strength in comparison to the manual method. Tis result was explained by the assumption that the mechanical method creates a less grainy mixture due to better water difusion [45]. Furthermore, the manual method causes insufcient hydration by restraining microchannel formation inside the MTA [38]. Te diference between the results of the abovementioned studies may be attributed to diferences in their methodologies. Uzunoglu et al. [45] did not include the ultrasonic method in the study; they investigated the efect of diferent moisture conditions on push-out bond strength, which was not investigated in the study by Shahi et al. [41]. Various brands of MTA used in two studies (ProRoot MTA vs. MTA Angelus) might also have an impact on the results.

Flexural Strength.
Te signifcance of enhanced fexural strength values in endodontic operations is that it helps the clinicians to use lower amounts of MTA. Tis feature is important where the space for material placement is limited, and the material should withstand occlusal loading or restorative procedures [78]. Te three-point bend test, which is a reliable and valid method, is usually used to evaluate fexural strength [79,80].
Basturk et al. [40] investigated the fexural strength of white MTA Angelus and white ProRoot MTA mixed with manual and mechanical methods. No signifcant diference was found between methods [40]. Since there is no data on the efects of the ultrasonic method on the fexural strength of MTA, further studies are needed to draw a defnitive conclusion.

Porosity.
Porosity is a measure of void spaces within a material. Tere is a negative correlation between the porosity and fexural strength of MTA [40]. On the other hand, porosity might be benefcial for the MTA hydration process because these pores may provide space for the water to penetrate the material [66].
Basturk et al. [40] investigated the porosity of two types of MTA Angelus and ProRoot MTA mixed with manual and mechanical methods using micro-CT at the end of the 4th day. In the second study, Sisli and Ozbas [47] investigated the porosity of two types of MTA Angelus and ProRoot MTA mixed with manual and mechanical methods using micro-CT at the end of the 7th day. Controversial results were observed concerning mechanical and manual methods, with Sisli and Ozbas [47] reporting higher porosity rates both within the material and at the MTA-dentin interface prepared with the manual method than the mechanical method [47]. Meanwhile, Basturk et al. [40] did not fnd any signifcant diferences between the same groups [40]. Tese contrasting results might be explained by diferent study designs and time of assessments.
In the third study, Ghasemi et al. [42] investigated the porosity of the MTA Angelus mixed with manual and ultrasonic methods using CBCT at the end of the 7 th day and reported that ultrasonic mixing results in lower void formation at the MTA-dentin interface than manual method due to the increased fow of the MTA [42]. Te increased fow of particles by the ultrasonic method can rearrange particles and displace the voids towards the surface releasing them from the mixture.
In summary, the lack of standard mixing and porosity assessment method makes it difcult to compare the results of diferent studies to draw a defnitive conclusion.

Hydration and Phase
Formation. X-ray difraction analysis is used to assess the hydration and phase formation of MTA. It works by detecting the interferences of monochromatic X-ray beams with the structures present in the material [81] and helps in detecting crystalline particles' formation, their transformations [6], and other various structural parameters [81].
Basturk et al. [46] investigated the hydration and phase formation of tooth-colored ProRoot MTA and White MTA Angelus mixed with manual and mechanical methods at the end of the 4 th day and reported that the highest amount of tricalcium silicate, dicalcium silicate, and calcium hydroxide formation in MTA Angelus samples was in those which were mechanically mixed and placed with ultrasonic activation as opposed to manual mixing. Tese particles are the main crystalline structures associated with MTA hydration [82,83]. However, they demonstrated no signifcant diferences among ProRoot MTA samples prepared by manual or mechanical methods [46]. Te diference between MTA Angelus and ProRoot MTA samples might be attributed to the more homogeneous chemical composition [84,85] and smaller particle sizes of ProRoot MTA samples resulting in a better wetting of the particles [86], and sample which is less dependent on various mixing methods to ensure hydration. In the second study, Saghiri et al. [43] investigated the hydration and phase formation of the White MTA mixed with manual, mechanical, and ultrasonic methods at three diferent time intervals (1, 7, and 21 days) and reported that the mechanical method resulted in the highest amount of calcium silicate phases followed by the manual and ultrasonic methods [43].
In summary, it seems that the mechanical method promotes crystallization and phase formation of calcium silicates within MTA by more thorough wetting of particles resulting in a better hydration [43,46]. Additionally, the mechanical technique prevents the clustering of the powder particles, resulting in more even distribution of particles [43]. Furthermore, direct ultrasonic mixing of the MTA samples can result in higher void formation, which prevents proper crystallization of MTA particles [43].

Sealing
Ability. MTA has an excellent sealing ability [9,10]. Studies have evaluated the efect of diferent parameters on the sealing ability of MTA [87][88][89]. One of the parameters which afect sealing ability is the mixing method.
Shahi and Ozbas [37] investigated the bacterial sealing ability of White MTA mixed with manual, mechanical, and ultrasonic methods within 120 days and showed that there was no signifcant diference in microleakage among the methods [37]. In the second study, Sisli and Ozbas [47] investigated the marginal adaptation of ProRoot MTA and MTA Angelus mixed with manual and mechanical mixing using micro-CT imaging on the 7 th day. Tey considered marginal adaptation as an indicator of sealing ability. Tey showed that the mechanical method improved the handling characteristics of the MTA, but there was no signifcant change in marginal adaptation [47]. Collectively, diferent mixing methods did not have a diferent efect on the sealing ability of MTA. 4.14. Limitations. No report of selection bias and varieties in methodologies were limitations of evidence. Limitations of this review were the lack of clinical trials about the subject which makes it hard to reach a fnal decision for the clinicians and the lack of studies for each physicochemical characteristic that hardens to defnitely interpret the reported results.

Conclusions
Considering the lack of sufcient studies and heterogeneity of experimental methods, the following conclusions could be made: (1) Ultrasonic mixing has a favorable efect on the MTA characteristics, including microhardness, fowability, solubility, setting time, and porosity. However, this technique might have an unfavorable efect on the hydration phase of MTA. (2) Mechanical mixing method showed favorable efects on some properties of MTA, including fowability, solubility, push-out bond strength, and the hydration. However, setting time might be adversely affected by this method. (3) Manual mixing method showed less favorable efects on microhardness, fowability, solubility, setting time, push-out bond strength, porosity, and hydration compared to mechanical and ultrasonic methods. (4) Finally, regarding the above-mentioned results and noticing that none of the three mixing methods had any superiority on such characteristics as compressive strength, sealing ability, pH and calcium ion release, volume change, flm thickness, and fexural strength, it seems that using the manual mixing method is not benefcial for achieving ideal physicochemical properties of MTA. Accordingly, ultrasonic and mechanical mixing methods may help clinicians to achieve satisfactory physicochemical properties. Nonetheless, further investigations are needed to reach more precise and reliable results.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that there are no conficts of interest.